Immunization with plasmid encoding immunogenic proteins and intracellular targeting sequences

ABSTRACT

Improved vaccines are disclosed. The improved vaccines include a nucleotide sequence that encodes a coding sequence that comprises an immunogenic target protein linked to or comprising an intracellular cellular targeting sequence, the coding sequence being operably linked to regulatory elements are disclosed. Methods of immunizing individuals are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 08/957,001 filed Oct. 23,1997, which claims priority to Provisional Application Ser. No.60/029,592 filed Oct. 23, 1996 which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to improved protective and therapeuticvaccines and improved methods for prophylactically and/ortherapeutically inducing immune responses against antigens.

BACKGROUND OF THE INVENTION

DNA vaccines represent an emerging field which provides the means toprevent and treat disorders, diseases, conditions and infections byinducing immune responses in individuals which are directed at antigensassociated with such disorders, diseases, conditions and infections.Essentially, plasmid DNA that includes coding sequences for antigensoperably linked to regulatory elements required for gene expression isadministered to individuals. The cells of the individual take up theplasmid DNA and the coding sequence is expressed. The antigen soproduced becomes a target against which an immune response is directed.The immune response directed against the antigen provided theprophylactic or therapeutic benefit to the individual against anyallergen, pathogen, cancer cell or autoimmune cell that includes anepitope that is recognized by the immune response against the antigen.

DNA vaccines include naked and facilitated vaccines. Further, they maybe administered by a variety of techniques including several differentdevices for administering substances to tissue. The published literatureincludes several review articles that describe aspects of DNA vaccinetechnology and cite some of the many reports of results obtained usingthe technology. The following review articles which are eachincorporated herein by reference as are each of the references cited ineach review article discuss DNA vaccine technology: McDonnel W. M and F.K. Askari 1996 New Engl. J. Med. 334(1)42-45; Robinson, A. 1995 Can.Med. Assoc. J. 152(10):1629-1632; Fynan, E. F. et al. 1995 Int. J.Immunopharmac. 17(2)79-83; Pardoll, D. M. and A. M. Beckerleg 1995Immunity 3:165-169; and Spooner et al. 1995 Gene Therapy 2:173-180.

While such vaccines are often effective to immunize individualsprophylactically or therapeutically against pathogen infection or humandiseases, there is a need for improved vaccines. There is a need forcompositions and methods which produce an enhanced immune response.

SUMMARY OF THE INVENTION

The present invention relates to a plasmid which comprises nucleotidesequences that encodes an immunogenic target antigen operably linked toregulatory elements necessary for expression in eukaryotic cells whereinthe nucleotide sequence that encodes the immunogenic antigen includes anucleotide sequence that encodes a signal sequence which directstrafficking of the immunogenic target antigen within the cell. In somepreferred embodiments, the immunogenic target antigen is a pathogenantigen, a cancer-associated antigen or an antigen linked to cellsassociated with autoimmune diseases. In some embodiments, the nucleotidesequence that encodes a signal sequence which directs trafficking of theimmunogenic target antigen within the cell encodes a signal sequencewhich directs the immunogenic target antigen to be secreted or tolocalize to the cytoplasm, the cell membrane, the endoplasmic reticulum,or a lysosome. In some embodiments, the nucleotide sequence that encodesa signal sequence which directs intracellular trafficking of theimmunogenic target antigen is a non-native signal sequence.

The present invention relates to a method of inducing, in an individual,an immune response against an antigen comprising the step ofadministering to an individual, a plasmid which comprises a nucleotidesequence that encodes an immunogenic target antigen operably linked toregulatory elements necessary for expression in eukaryotic cells whereinthe nucleotide sequence that encodes the immunogenic antigen includes anucleotide sequence that encodes a signal sequence which directstrafficking of the immunogenic target antigen within the cell. In somepreferred embodiments, the immunogenic target antigen is a pathogenantigen, a cancer-associated antigen or an antigen linked to cellsassociated with autoimmune diseases. In some embodiments, the nucleotidesequence that encodes a signal sequence which directs trafficking of theimmunogenic target antigen within the cell encodes a signal sequencewhich directs the immunogenic target antigen to be secreted or tolocalize to the cytoplasm, the cell membrane, the endoplasmic reticulum,or a lysosome. In some embodiments, the nucleotide sequence that encodesa signal sequence which directs intracellular trafficking of theimmunogenic target antigen is a non-native signal sequence.

The present invention relates to improved DNA vaccines which comprisesnucleotide sequences that encodes an immunogenic target antigen operablylinked to regulatory elements necessary for expression in eukaryoticcells wherein the nucleotide sequence that encodes the immunogenicantigen includes a nucleotide sequence that encodes a signal sequencewhich directs trafficking of the immunogenic target antigen within thecell. In some preferred embodiments, the immunogenic target antigen is apathogen antigen, a cancer-associated antigen or an antigen linked tocells associated with autoimmune diseases. In some embodiments, thenucleotide sequence that encodes a signal sequence which directstrafficking of the immunogenic target antigen within the cell encodes asignal sequence which directs the immunogenic target antigen to besecreted or to localize to the cytoplasm, the cell membrane, theendoplasmic reticulum, or a lysosome. In some embodiments, thenucleotide sequence that encodes a signal sequence which directsintracellular trafficking of the immunogenic target antigen is anon-native signal sequence.

The present invention relates to a method of immunizing an individualagainst a pathogen, cancer or an autoimmune disease comprising the stepof administering to an individual, a DNA vaccine which comprises anucleotide sequence that encodes an immunogenic target antigen operablylinked to regulatory elements necessary for expression in eukaryoticcells wherein the nucleotide sequence that encodes the immunogenicantigen includes a nucleotide sequence that encodes a signal sequencewhich directs trafficking of the immunogenic target antigen within thecell. In some preferred embodiments, the immunogenic target antigen is apathogen antigen, a cancer-associated antigen or an antigen linked tocells associated with autoimmune diseases. In some embodiments, thenucleotide sequence that encodes a signal sequence which directstrafficking of the immunogenic target antigen within the cell encodes asignal sequence which directs the immunogenic target antigen to besecreted or to localize to the cytoplasm, the cell membrane, theendoplasmic reticulum, or a lysosome. In some embodiments, thenucleotide sequence that encodes a signal sequence which directsintracellular trafficking of the immunogenic target antigen is anon-native signal sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data from FACS analysis of H221 binding.

FIG. 2A shows a map of the genetic immunization vector into which the VHor Fv regions were cloned.

FIG. 2B shows a map of the constructions of inserts that encode the Vregions used to target the V regions to different locations in the cell.

FIG. 3 shows results of experiments in which the different constructdesigned to be targeted at various locations within the cell werecompared for their induction of cytotoxic T cell and proliferativeresponses.

FIGS. 4A, 4B and 4C show results from experiments evaluating CTLresponses elicited by various intracellular targeted DNA vaccines.

FIGS. 5A, 5B and 5C show results from tumor challenge experiments usinghybridoma cells that produce the antibody whose variable region isencoded by the DNA vaccine.

FIGS. 6A and 6B show genetic constructs of the invention that comprisespecific leader-sequences.

FIGS. 7A and 7B shows several C terminal sequences for ER retention.

FIG. 8 shows the structure of DNA Vaccines. The DNA Vaccine backboneused was the pBBkan backbone. This uses the CMV promoter and RSVenhancer to drive transcription. The inserts are shown in Table 4, withV_(H) Fv (V_(L) linked to V_(H)) regions following a leader peptide(either a hydrophobic leader from murine IgG (Ig Leader), or ahydrophilic leader for cytosolic targeting (Cyto Leader)), and an addedtransmembrane and cytosolic tail with an endoplasmic reticulum retentionsignal (CD4 TM & E19 Cyto).

FIG. 9 shows results of experiments to evaluate the proliferativeresponse of spleenocytes following a single DNA inoculation.

FIGS. 10A, 10B and 10C show results of experiments to evaluate thecytotoxic T cell (CTL) response following a single DNA inoculation. FIG.10A shows data from experiments in which the vaccines targeted the H221V_(H) or Fv region to the ER for secretion. FIG. 10B shows data fromexperiments in which the vaccines targeted the H221 V_(H) or Fv regionto the ER for retention. FIG. 10C shows data from experiments in whichthe vaccines targeted the H221 V_(H) or Fv region to the cytosol.

FIGS. 11A and 11B show data regarding survival and tumor burdenfollowing DNA inoculation and challenge.

FIG. 12 shows data form experiments evaluating the proliferativeresponse in survivors of tumor challenge.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved DNA vaccines. DNA vaccines aredescribed in U.S. Pat. Nos. 5,589,466 and 5,973,972, and PCT publishedapplications PCT/US90/01515, PCT/US93/02338, PCT/US93/048131, andPCT/US94/00899, and the priority applications cited therein, which areeach incorporated herein by reference. In addition to the deliveryprotocols described in those applications, alternative methods ofdelivering DNA are described in U.S. Pat. Nos. 4,945,050 and 5,036,006,which are both incorporated herein by reference. Further, the reviewarticles cited above describe DNA vaccine technology and cite examplesof DNA vaccines. In each case, plasmid DNA is delivered to cells of anindividual which take up the plasmid and express immunogenic targetproteins encoded by the plasmids. The immune response generated againstthe immunogenic target protein provides a prophylactic or therapeuticbenefit to the vaccinated individual.

According to the present invention, the coding sequence on the plasmidthat encodes the immunogenic target protein is provided with a codingsequence that encodes an amino acid sequence whose presence on theprotein results in a specific intracellular localization of theexpressed protein. The nucleotide sequences that encode amino acidsequences which direct intracellular protein trafficking and which areincluded in the coding sequences of immunogenic proteins that areincluded in plasmid constructs used as DNA vaccine compositions directlocalization to specific areas in the cells which result in enhancementof specific immune responses.

As used herein, the term “genetic construct” is meant to refer toplasmids which comprise coding sequences that encode an immunogenictarget protein and an amino acid sequence that directs intracellularprotein trafficking, the coding sequences being operably linked toregulatory elements required for expression of the coding sequences ineukaryotic cells. Regulatory elements for DNA expression include apromoter and a polyadenylation signal. In addition, other elements, suchas a Kozak region, may also be included in the genetic construct.Initiation and termination signals are required regulatory elementswhich are often considered part of the coding sequence. The codingsequences of genetic constructs of the invention include functionalinitiation and termination signals.

As used herein, the term “immunogenic target protein” is meant to referto an antigen that is a target for an immune response which is directedat proteins associated with conditions, infections, diseases ordisorders such as allergens, pathogen antigens, antigens associated withcancer cells or cells involved in autoimmune diseases. The immunogenictarget antigen is encoded by the coding sequence of a genetic constructused in a DNA vaccine. The DNA vaccine is administered to the vaccinatedindividual, the genetic construct is taken up by the cells of theindividual, the coding sequence is expressed and the immunogenic targetprotein is produced. The immunogenic target protein induces an immuneresponse against the immunogenic target protein in the individual. Theimmune response is directed against proteins associated with conditions,infections, diseases or disorders such as allergens, pathogen antigens,antigens associated with cancer cells or cells involved in autoimmunediseases. Thus the vaccinated individual may be immunizedprophylactically or therapeutically to prevent or treat conditions,infections, diseases or disorders. The immunogenic target protein refersto peptides and protein encoded by gene constructs of the presentinvention which act as target proteins for an immune response. The term“immunogenic target protein” refers to a protein against which an immuneresponse can be elicited. The immunogenic target protein shares at leastan epitope with a protein from the allergen, pathogen or undesirableprotein or cell-type such as a cancer cell or a cell involved inautoimmune disease against which immunization is required. The immuneresponse directed against the immunogenic target protein will protectthe individual against and treat the individual for the specificinfection or disease with which the protein from the allergen, pathogenor undesirable protein or cell-type is associated. The immunogenictarget protein does not need to be identical to the protein againstwhich an immune response is desired. Rather, the immunogenic targetprotein must be capable of inducing an immune response that cross reactsto the protein against which the immune response is desired.

As used herein, the term “non-native signal sequence” is meant to referto signal sequences that are heterologous with respect to the nucleotidesequence that encodes a signal sequence which directs intracellulartrafficking of the immunogenic target antigen. A non-native signalsequence is not found linked to the immunogenic target protein in naturebut rather is brought about by preparing a gene construct in which thenucleotide sequence that encodes a signal sequence is linked with anucleotide sequence that encodes the immunogenic target antigen. In someembodiments, a native signal sequence may be removed from a codingsequence that encodes an immunogenic target protein and replaced with anon-native signal sequence to direct the localization of the protein toa location different from the location that the native sequences directsproteins to or to more efficiently direct localization to the samelocation that the native signal sequence directs localization to.

According to the present invention, the immunogenic target proteinincludes sequences that direct its localization within the cell. Thenaturally occurring sequences that direct protein localization may beincorporated into immunogenic target proteins of DNA vaccines byproviding signal sequences, designing chimeric proteins or grafting thesequence into the immunogenic protein sequence. The DNA vaccines areplasmids and the coding sequences of the immunogenic target proteins aremanipulated by standard molecular biology methodology to produce codingsequences that encode immunogenic target proteins which include signalsequences that direct intracellular protein trafficking, or chimericimmunogenic target proteins that include regions which directintracellular protein trafficking. Moreover, routine molecular biologytechniques may be employed to change the amino acid sequence of animmunogenic target protein so that it contains within its sequence thesequences that direct intracellular protein targeting. In someembodiments, the nucleotide sequence that encodes a signal sequencewhich directs intracellular trafficking of the immunogenic targetantigen is a non-native signal sequence. The nucleotide sequence thatencodes a signal sequence of one protein may be identified, isolated andlinked to a coding sequence that encodes a different protein using wellknown techniques.

The location wherein the cell the immunogenic target protein is directedaffects the immune response generated by the individual against theimmunogenic target protein. It has been discovered that theintracellular targeting of immunogenic target proteins encoded bygenetic constructs of DNA vaccines results in an enhanced immuneresponse against the immunogenic target antigen. By providing codingsequence of the intracellular targeting signal as part of the codingsequence of the immunogenic target protein, the immunogenic targetprotein is localized to a part of the cell. It has been discovered thatcertain localizations are associated with enhanced specific types ofimmune responses. For example, it has been discovered that directingprotein to be retained or recycled to the endoplasmic reticulum,particularly the rough endoplasmic reticulum results in induction of anenhance CTL response in vaccinated animals relative to that observedusing vaccines that do not include sequences that target specificintracellular localization.

The improvement of the present invention relates to the inclusion ofgenetic material for directing the intracellular localization ofimmunogenic target proteins produced in cells of individualsadministered a DNA vaccine.

The present invention relates to methods of introducing genetic materialinto the cells of an individual in order to induce immune responsesagainst proteins and peptides which are encoded by the genetic material.The methods comprise the steps of administering to the tissue of saidindividual, DNA that includes a coding sequence operably linked toregulatory elements required for expression. The coding sequenceincludes coding sequences for immunogenic target proteins linked to orcomprising a coding sequence for an intracellular trafficking signal.

Intracellular trafficking signals are well known.

In some embodiments, proteins are to be secreted. Such proteins includean N-terminal hydrophobic sequence. When RNA is translated, thehydrophobic sequence at the N terminal causes the protein to stick tothe rough endoplasmic reticulum. The hydrophobic sequences aresubsequently clipped off by a protease and the protein is secreted. Insome embodiments of the present invention, the immunogenic targetprotein may include an N terminal hydrophobic leader sequence which willdirect secretion of the immunogenic target protein when expressed in thecell.

In some embodiments, proteins are to be membrane bound. Such proteinsinclude an N-terminal hydrophobic sequence and an internal hydrophobicregion. As in the secreted forms, when RNA is translated, thehydrophobic sequences causes the protein to stick to the roughendoplasmic reticulum. The N terminal hydrophobic sequence issubsequently clipped off by a protease. The protein follows the samesecretion pathway but the internal hydrophobic sequence preventssecretion and the protein becomes membrane bound. In some embodiments ofthe present invention, the immunogenic target protein may include an Nterminal hydrophobic leader sequence and an internal hydrophobicsequence which will result in the immunogenic target protein becoming amembrane bound protein when expressed in the cell.

In some embodiments, proteins are to be localized in the cytosol. Suchproteins do not have an N-terminal hydrophobic sequence. When RNA istranslated, the protein does not stick to the rough endoplasmicreticulum and the protein becomes cytosolic. In some embodiments of thepresent invention, the immunogenic target protein is free of an Nterminal hydrophobic leader sequence so that the immunogenic targetprotein becomes a cytosolic protein when expressed in the cell.

In some embodiments, proteins are to be localized in the lysosome. Suchproteins may include the sequence DKQTLL (SEQ ID NO:1) which directslocalization of proteins to the lysosome. In some embodiments of thepresent invention, the immunogenic target protein includes the sequenceDKQTLL (SEQ ID NO:1) so that the immunogenic target protein is directedto the lysosome when expressed in the cell.

In some embodiments, proteins are to be localized from the Golgi bodyback to the ER. Such proteins may include the sequence KDEL (SEQ IDNO:2) at the C terminal which directs localization of proteins to berecycled to the ER. In some embodiments of the present invention, theimmunogenic target protein includes the sequence KDEL (SEQ ID NO:2) atthe C terminal so that the immunogenic target protein is directed to theER.

Another example of such an “ER recycling signal” is reported to be the Cterminal sequence of the E19 protein from adenovirus. That protein islocalized to the ER where it binds to the MHCs and effectively keepsthem from loading proteins which are presented by the MHC at the surfacewhere they complex with T cell receptors as part of immune responseinduction. The E109 protein is a hexapeptide DEKKMP (SEQ ID NO:3). Insome embodiments, proteins are to be localized to the ER by includingthe DEKKMP (SEQ ID NO:3) sequence at the C terminal. In some embodimentsof the present invention, the immunogenic target protein includes thesequence DEKKMP (SEQ ID NO:3) at the C terminal so that the immunogenictarget protein is directed to the ER.

Depending upon the type of immune response sought to be enhanced,different intracellular localization is desirable. In the case of ClassI immune responses, proteins synthesized within a cell are degraded andtransported into the ER where they are loaded onto MHCs which then moveto the cell surface and complex with T cell receptors of CD8⁺T cells.This action leads to CTL responses. In the case of Class II immuneresponses, proteins are complexed with antigen presenting cells (APCs)which interact with CD4⁺T cells, engaging helper T cells including thoseassociated with antibody responses.

In order to enhance Class I immune responses, localization of proteinsto the cytosol or ER allows for such proteins to be more accessible tothe Class I pathway.

In order to enhance Class II immune responses, localization of proteinsto the transmembrane or lysosomes, or secretion of the protein allowssuch proteins to be more accessible to the Class II pathway.

The present invention provides genetic constructs useful as DNA vaccinesthat include coding sequences for immunogenic target proteins thatcomprise sequences for intracellular localization.

In some embodiments, genetic constructs comprise the localizationleaders described in Biocca, S. et al. 1990 EMBO J. 9:101-108. Thegenetic constructs contain those leader sequences in the sameorientation as they are described in FIG. 6A. Specifically, the leadersequences occur at the N terminus of the protein directly between thepromoter and coding sequence of the protein of interest. In someembodiments, the genetic constructs of the invention comprise one of theleader sequences set forth in FIG. 6A of this application. In someembodiments, the genetic constructs of the invention comprise one of theleader sequences set forth in FIG. 6B of this application.

In some embodiments, genetic constructs comprise the terminalhexapeptide from adenovirus E19 protein for retention in the endoplasmicreticulum. When the C terminus of an immunogenic target protein of a DNAvaccine is DEKKMP (SEQ ID NO:3), the protein will be retained in the ER.

In some embodiments, genetic constructs comprise the C terminalquadrapeptide for retention in the endoplasmic reticulum. When the Cterminus of an immunogenic target protein of a DNA vaccine is KDEL (SEQID NO:2), the protein will be retained in the ER.

FIGS. 7A and 7B shows several C terminal sequences for ER retention.Jackson, M. R., et al. 1990 EMBO J. 9:3153-3162 reports that C terminalsequences in FIG. 7A, when linked at the C terminal of proteins normallynot retained in the ER, results in ER retention of the chimeric protein.

In some embodiments, genetic constructs comprise the lysosomal targetingdoublets at the C terminal tail of the immunogenic target protein. The Cterminal tail is the last 20-30 amino acids. By including the doubletsLL and/or YQ and/or QY the protein is directed to a lysosome. In someembodiments, the C terminal tail which contains one or more lysosometargeting doublets is a cytoplasmic tail of a transmembrane protein. Insome embodiments, the doublets are included in the sequence of theimmunogenic protein within the last 30 amino acids.

According to the present invention, compositions and methods areprovided which prophylactically and/or therapeutically immunize anindividual against allergens, pathogens or abnormal, disease-relatedcells or proteins. The genetic material that encodes a target protein,i.e. a peptide or protein that shares at least an epitope with animmunogenic protein found on the allergen, pathogen or antigen or cellto be targeted, and genetic material that encodes an intracellulartrafficking signal. The genetic material is expressed by theindividual's cells and serves as an immunogenic target against which animmune response is elicited. The presence of the intracellulartrafficking signal directs the localization of the protein to a locationin the cell where it is more effective in inducing a desired immuneresponse. The resulting immune response that reacts with the allergen,pathogen or antigens or cells is enhanced relative to the immuneresponse induced by similar genetic constructs which lack theintracellular trafficking signal.

The present invention is useful to elicit immune responses againstproteins specifically associated with allergies, pathogens or theindividual's own “abnormal” cells. The present invention is useful toimmunize individuals against allergens or pathogenic agents andorganisms such that an immune response against a pathogen proteinprovides protective immunity against the pathogen. The present inventionis useful to combat hyperproliferative diseases and disorders such ascancer by eliciting an immune response against a target protein that isspecifically associated with the hyperproliferative cells. The presentinvention is useful to combat autoimmune diseases and disorders byeliciting an immune response against a target protein that isspecifically associated with cells involved in the autoimmune condition.

According to the present invention, DNA encodes an immunogenic targetprotein that is either linked to an intracellular trafficking signalsequence or a peptide sequence that includes an intracellulartrafficking sequence. A signal sequence will be clipped off as part ofthe cells normal protein processing. In addition, DNA that encodes animmunogenic target protein may include, within the coding sequence ofthe protein, an intracellular trafficking sequence. Regulatory elementsfor DNA expression include a promoter and a polyadenylation signal. Inaddition, other elements, such as a Kozak region, may also be includedin the genetic construct.

As used herein, the term “expressible form” refers to gene constructswhich contain the necessary regulatory elements operably linked to acoding sequence that encodes an immunogenic target protein such thatwhen present in the cell of the individual, the coding sequence will beexpressed.

As used herein, the term “sharing an epitope” refers to proteins whichcomprise at least one epitope that is identical to or substantiallysimilar to an epitope of another protein.

As used herein, the term “substantially similar epitope” is meant torefer to an epitope that has a structure which is not identical to anepitope of a protein but nonetheless invokes a cellular or humoralimmune response which cross reacts to that protein.

Genetic constructs comprise a nucleotide sequence that encodes animmunogenic target protein that includes an intracellular traffickingsequence operably linked to regulatory elements needed for geneexpression.

When taken up by a cell, the genetic construct(s) may remain present inthe cell as a functioning extrachromosomal molecule and/or integrateinto the cell's chromosomal DNA. DNA may be introduced into cells whereit remains as separate genetic material in the form of a plasmid orplasmids. Alternatively, linear DNA which can integrate into thechromosome may be introduced into the cell. When introducing DNA intothe cell, reagents which promote DNA integration into chromosomes may beadded. DNA sequences which are useful to promote integration may also beincluded in the DNA molecule. Alternatively, RNA may be administered tothe cell. It is also contemplated to provide the genetic construct as alinear minichromosome including a centromere, telomeres and an origin ofreplication. Gene constructs may remain part of the genetic material inattenuated live microorganisms or recombinant microbial vectors whichlive in cells. Gene constructs may be part of genomes of recombinantviral vaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal.

Genetic constructs include regulatory elements necessary for geneexpression of a nucleic acid molecule. The elements include: a promoter,an initiation codon, a stop codon, and a polyadenylation signal. Inaddition, enhancers are often required for gene expression of thesequence that encodes the immunogenic target protein. It is necessarythat these elements be operably linked to the sequence that encodes thedesired proteins and that the regulatory elements are operable in theindividual to whom they are administered.

Initiation codons and stop codons are generally considered to be part ofa nucleotide sequence that encodes the immunogenic target protein.However, it is necessary that these elements are functional in theindividual to whom the gene construct is administered. The initiationand termination codons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to: human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs can be provided with mammalian origin of replicationin order to maintain the construct extrachromosomally and producemultiple copies of the construct in the cell. Plasmids pCEP4 and pREP4from Invitrogen (San Diego, Calif.) contain the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region whichproduces high copy episomal replication without integration.

In some preferred embodiments related to immunization applications,nucleic acid molecule(s) are delivered which include nucleotidesequences that encode a target protein, IL-12 protein and, additionally,genes for proteins which further enhance the immune response againstsuch target proteins. Examples of such genes are those which encodecytokines and lymphokines such as α-interferon, gamma-interferon,platelet derived growth factor (PDGF), GC-SF, GM-CSF, TNF, epidermalgrowth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and B7.2. Insome embodiments, it is preferred that the gene for GM-CSF is includedin genetic constructs used in immunizing compositions.

An additional element may be added which serves as a target for celldestruction if it is desirable to eliminate cells receiving the geneticconstruct for any reason. A herpes thymidine kinase (tk) gene in anexpressible form can be included in the genetic construct. The druggangcyclovir can be administered to the individual and that drug willcause the selective killing of any cell producing tk, thus, providingthe means for the selective destruction of cells with the geneticconstruct.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce DNA constructs which are functional in the cells.

The method of the present invention comprises the steps of administeringnucleic acid molecules to tissue of the individual. In some preferredembodiments, the nucleic acid molecules are administeredintramuscularly, intranasally, intraperatoneally, subcutaneously,intradermally, or topically or by lavage to mucosal tissue selected fromthe group consisting of vaginal, rectal, urethral, buccal andsublingual.

In some embodiments, the nucleic acid molecule is delivered to the cellsin conjunction with administration of a facilitating agent. Facilitatingagents are also referred to as polynucleotide function enhancers orgenetic vaccine facilitator agents. Facilitating agents are described inU.S. Ser. No. 08/008,342 filed Jan. 26, 1993, U.S. Ser. No. 08/029,336filed Mar. 11, 1993, U.S. Ser. No. 08/125,012 filed Sep. 21, 1993, andInternational Application Ser. No. PCT/US94/00899 filed Jan. 26, 1994,which are each incorporated herein by reference. In addition,facilitating agents are described in PCT application Ser. No.PCT/US95/04071 filed Mar. 30, 1995, which is incorporated herein byreference. Facilitating agents which are administered in conjunctionwith nucleic acid molecules may be administered as a mixture with thenucleic acid molecule or administered separately simultaneously, beforeor after administration of nucleic acid molecules. In addition, otheragents which may function transfecting agents and/or replicating agentsand/or inflammatory agents and which may be co-administered with orwithout a facilitating agent include growth factors, cytokines andlymphokines such as α-interferon, gamma-interferon, platelet derivedgrowth factor (PDGF), GC-SF, GM-CSF, TNF, epidermal growth factor (EGF),IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and B7.2 as well as fibroblastgrowth factor, surface active agents such as immune-stimulatingcomplexes (ISCOMS), Freund's incomplete adjuvant, LPS analog includingmonophosphoryl Lipid A (MPL), muramyl peptides, quinone analogs andvesicles such as squalene and squalene, and hyaluronic acid may also beadministered in conjunction with the genetic construct.

In some preferred embodiments, the genetic constructs of the inventionare formulated with or administered in conjunction with a facilitatorselected from the group consisting of benzoic acid esters, anilides,amidines, urethans and the hydrochloride salts thereof such as those ofthe family of local anesthetics.

The facilitator in some preferred embodiments may be a compound havingone of the following formulae:

Ar—R¹—O—R²—R³

or

Ar—N—R¹—R²—R³

or

R⁴—N—R⁵—R⁶

or

R⁴—O—R¹—N—R⁷

wherein:

Ar is benzene, p-aminobenzene, m-aminobenzene, o-aminobenzene,substituted benzene, substituted p-aminobenzene, substitutedm-aminobenzene, substituted o-aminobenzene, wherein the amino group inthe aminobenzene compounds can be amino, C₁-C₅ alkylamine, C₁-C₅, C₁-C₅dialkylamine and substitutions in substituted compounds are halogen,C₁-C₅ alkyl and C₁-C₅ alkoxy;

R¹ is C═O;

R² is C₁-C₁₀ alkyl including branched alkyls;

R³ is hydrogen, amine, C₁-C₅ alkylamine, C₁-C₅, C₁-C₅ dialkylamine;

R²+R³ can form a cyclic alkyl, a C₁-C₁₀ alkyl substituted cyclic alkyl,a cyclic aliphatic amine, a C₁-C₁₀ alkyl substituted cyclic aliphaticamine, a heterocycle, a C₁-C₁₀ alkyl substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle;

R⁴ is Ar, R² or C₁-C₅ alkoxy, a cyclic alkyl,a C₁-C₁₀ alkyl substitutedcyclic alkyl, a cyclic aliphatic amine, a C₁-C₁₀ alkyl substitutedcyclic aliphatic amine, a heterocycle, a C₁-C₁₀ alkyl substitutedheterocycle and a C₁-C₁₀ alkoxy substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle;

R⁵ is C═NH;

R⁶ is Ar, R² or C₁-C₅ alkoxy, a cyclic alkyl,a C₁-C₁₀ alkyl substitutedcyclic alkyl, a cyclic aliphatic amine, a C₁-C₁₀ alkyl substitutedcyclic aliphatic amine, a heterocycle, a C₁-C₁₀ alkyl substitutedheterocycle and a C₁-C₁₀ alkoxy substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle; and.

R⁷ is Ar, R² or C₁-C₁₀ alkoxy, a cyclic alkyl,a C₁-C₁₀ alkyl substitutedcyclic alkyl, a cyclic aliphatic amine, a C₁-C₁₀ alkyl substitutedcyclic aliphatic amine, a heterocycle, a C₁-C₁₀ alkyl substitutedheterocycle and a C₁-C₁₀ alkoxy substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle.

Examples of esters include: benzoic acid esters such as piperocaine,meprylcaine and isobucaine; para-aminobenzoic acid esters such asprocaine, tetracaine, butethamine, propoxycaine and chloroprocaine;meta-aminobenzoic acid esters including metabuthamine and primacaine;and para-ethoxybenzoic acid esters such as parethoxycaine. Examples ofanilides include lidocaine, etidocaine, mepivacaine, bupivacaine,pyrrocaine and prilocaine. Other examples of such compounds includedibucaine, benzocaine, dyclonine, pramoxine, proparacaine, butacaine,benoxinate, carbocaine, methyl bupivacaine, butasin picrate, phenacaine,diothan, luccaine, intracaine, nupercaine, metabutoxycaine, piridocaine,biphenamine and the botanically-derived bicyclics such as cocaine,cinnamoylcocaine, truxilline and cocaethylene and all such compoundscomplexed with hydrochloride.

In preferred embodiments, the facilitator is bupivacaine. The differencebetween bupivacaine and mepivacaine is that bupivacaine has a N-butylgroup in place of an N-methyl group of mepivacaine. Compounds may haveat that N, C₁-C₁₀. Compounds may be substituted by halogen such asprocaine and chloroprocaine. The anilides are preferred.

The facilitating agent is administered prior to, simultaneously with orsubsequent to the genetic construct. The facilitating agent and thegenetic construct may be formulated in the same composition.

Bupivacaine-HCl is chemically designated as 2-piperidinecarboxamide,1-butyl-N-(2,6-dimethylphenyl)-monohydrochloride, monohydrate and iswidely available commercially for pharmaceutical uses from many sourcesincluding from Astra Pharmaceutical Products Inc. (Westboro, Mass.) andSanofi Winthrop Pharmaceuticals (New York, N.Y.), Eastman Kodak(Rochester, N.Y.). Bupivacaine is commercially formulated with andwithout methylparaben and with or without epinephrine. Any suchformulation may be used. It is commercially available for pharmaceuticaluse in concentration of 0.25%, 0.5% and 0.75% which may be used on theinvention. Alternative concentrations, particularly those between 0.05%-1.0% which elicit desirable effects may be prepared if desired.According to the present invention, about 250 μg to about 10 mg ofbupivacaine is administered. In some embodiments, about 250 μg to about7.5 mg is administered. In some embodiments, about 0.05 mg to about 5.0mg is administered. In some embodiments, about 0.5 mg to about 3.0 mg isadministered. In some embodiments about 5 to 50 μg is administered. Forexample, in some embodiments about 50 μl to about 2 ml, preferably 50 μlto about 1500 μl and more preferably about 1 ml of 0.25-0.50%bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceuticalcarrier is administered at the same site as the vaccine before,simultaneously with or after the vaccine is administered. Similarly, insome embodiments, about 50 μl to about 2 ml, preferably 50 μl to about1500 μl and more preferably about 1 ml of 0.25-0.50% bupivacaine-HCl inan isotonic pharmaceutical carrier is administered at the same site asthe vaccine before, simultaneously with or after the vaccine isadministered. Bupivacaine and any other similarly acting compounds,particularly those of the related family of local anesthetics may beadministered at concentrations which provide the desired facilitation ofuptake of genetic constructs by cells.

In some embodiments of the invention, the individual is first subject toinjection of the facilitator prior to administration of the geneticconstruct. That is, for example, up to a about a week to ten days priorto administration of the genetic construct, the individual is firstinjected with the facilitator. In some embodiments, the individual isinjected with the facilitator about 1 to 5 days; in some embodiments 24hours, before or after administration of the genetic construct.Alternatively, if used at all, the facilitator is administeredsimultaneously, minutes before or after administration of the geneticconstruct. Accordingly, the facilitator and the genetic construct may becombined to form a single pharmaceutical composition.

In some embodiments, the genetic constructs are administered free offacilitating agents, that is in formulations free from facilitatingagents using administration protocols in which the genetic constructionsare not administered in conjunction with the administration offacilitating agents.

Nucleic acid molecules which are delivered to cells according to theinvention may serve as genetic templates for proteins that function asprophylactic and/or therapeutic immunizing agents. In preferredembodiments, the nucleic acid molecules comprise the necessaryregulatory sequences for transcription and translation of the codingregion in the cells of the animal.

The present invention may be used to immunize an individual against allpathogens such as viruses, prokaryote and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites. The present invention is particularly useful to immunize anindividual against those pathogens which infect cells and which are notencapsulated such as viruses, and prokaryote such as gonorrhea, listeriaand shigella. In addition, the present invention is also useful toimmunize an individual against protozoan pathogens which include a stagein the life cycle where they are intracellular pathogens. As usedherein, the term “intracellular pathogen” is meant to refer to a virusor pathogenic organism that, at least part of its reproductive or lifecycle, exists within a host cell and therein produces or causes to beproduced, pathogen proteins. Table 1 provides a listing of some of theviral families and genera for which vaccines according to the presentinvention can be made. DNA constructs that comprise DNA sequences whichencode the peptides that comprise at least an epitope identical orsubstantially similar to an epitope displayed on a pathogen antigen suchas those antigens listed on the tables are useful in vaccines. Moreover,the present invention is also useful to immunize an individual againstother pathogens including prokaryotic and eukaryotic protozoan pathogensas well as multicellular parasites such as those listed on Table 2.

In order to produce a genetic vaccine to protect against pathogeninfection, genetic material which encodes immunogenic proteins againstwhich a protective immune response can be mounted must be included in agenetic construct as the coding sequence for the target. Whether thepathogen infects intracellularly, for which the present invention isparticularly useful, or extracellularly, it is unlikely that allpathogen antigens will elicit a protective response. Because DNA and RNAare both relatively small and can be produced relatively easily, thepresent invention provides the additional advantage of allowing forvaccination with multiple pathogen antigens. The genetic construct usedin the genetic vaccine can include genetic material which encodes manypathogen antigens. For example, several viral genes may be included in asingle construct thereby providing multiple targets.

Tables 1 and 2 include lists of some of the pathogenic agents andorganisms for which genetic vaccines can be prepared to protect anindividual from infection by them. In some preferred embodiments, themethods of immunizing an individual against a pathogen are directedagainst HIV, HTLV or HBV.

Another aspect of the present invention provides a method of conferringa broad based protective immune response against hyperproliferatingcells that are characteristic in hyperproliferative diseases and to amethod of treating individuals suffering from hyperproliferativediseases. As used herein, the term “hyperproliferative diseases” ismeant to refer to those diseases and disorders characterized byhyperproliferation of cells. Examples of hyperproliferative diseasesinclude all forms of cancer and psoriasis.

It has been discovered that introduction of a genetic construct thatincludes a nucleotide sequence which encodes an immunogenic“hyperproliferating cell”-associated protein into the cells of anindividual results in the production of those proteins in the vaccinatedcells of an individual. As used herein, the term“hyperproliferative-associated protein” is meant to refer to proteinsthat are associated with a hyperproliferative disease. To immunizeagainst hyperproliferative diseases, a genetic construct that includes anucleotide sequence which encodes a protein that is associated with ahyperproliferative disease is administered to an individual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein which is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk andEGRF. In addition to oncogene products as target antigens, targetproteins for anti-cancer treatments and protective regimens includevariable regions of antibodies made by B cell lymphomas and variableregions of T cell receptors of T cell lymphomas which, in someembodiments, are also used to target antigens for autoimmune disease.Other tumor-associated proteins can be used as target proteins such asproteins which are found at higher levels in tumor cells including theprotein recognized by monoclonal antibody 17-1A and folate bindingproteins.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andtechnology as well as epidemiology allow for the determination ofprobability and risk assessment for the development of cancer in theindividual. Using genetic screening and/or family health histories, itis possible to predict the probability a particular individual fordeveloping any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had a type ofcancer and is at risk of a relapse, they can be immunized in order toprepare their immune system to combat any future appearance of thecancer.

The present invention provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of genetic constructs serves as an immunotherapeutic,directing and promoting the immune system of the individual to combathyperproliferative cells that produce the target protein.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity including cell receptors and cells which produce“self”-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ-14, Vβ-17 and Vα-17. Thus, vaccination with a DNAconstruct that encodes at least one of these proteins will elicit animmune response that will target T cells involved in RA. See: Howell,M.D., et al., 1991 Proc. Natl. Acad. Sci. USA 88:10921-10925; Paliard,X., et al., 1991 Science 253:325-329; Williams, W. V., et al., 1992 J.Clin. Invest. 90:326-333; each of which is incorporated herein byreference.

In MS, several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include Vβ-7 and Vα-10.Thus, vaccination with a DNA construct that encodes at least one ofthese proteins will elicit an immune response that will target T cellsinvolved in MS. See: Wucherpfennig, K. W., et al., 1990 Science248:1016-1019; Oksenberg, J. R., et al., 1990 Nature 345:344-346; eachof which is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 andVα-12. Thus, vaccination with a DNA construct that encodes at least oneof these proteins will elicit an immune response that will target Tcells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines can be preparedusing this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes DNA constructsthat encode the variable region of such anti-DNA antibodies found in thesera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. 1987 Sequence off Proteins offimmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci.USA 87:1066, which is incorporated herein by reference.

EXAMPLES Example 1

Nephritogenic Autoantibody Anti-DNA IL/IM

This mAb was identified from a large panel of hybridomas derived fromMRL-lpr/lpr mice (Vlahakos, et al., 1992 Kidney Int 41,1690-1700)because it shared antigen binding properties with Ig eluted from thekidneys of MRL-lpr/lpr mice with nephritis. Anti-DNA IL/IM is an IgG2aantibody of the J558 VH gene family with a pI of 5.1. Anti-DNA IL/IM Abspecifically binds to ssDNA, dsDNA, SmRNP, and glomerular extract.Following administration to normal, histocompatible mice, anti-DNA IL/IMforms mesangial, subendothelial and intraluminal immune deposits in thekidneys. When anti-DNA IL/IM producing hybridoma cells are administeredintraperitoneally to histocompatible mice, they produce denseintramembranous and intraluminal deposits, associated with capillarywall thickening, mesangial interposition and expansion, aneurysmaldilatation and intraluminal occlusion of glomerular capillary loops, andheavy proteinuria. Although the morphologic appearance of the glomerularimmune deposits are reminiscent of those associated withcryoglobulinemia, anti-DNA IL/IM hybridoma-bearing mice with high serumanti-DNA Ab activity did not have detectable cryoglobulins or rheumatoidfactor activity. Glomerular immune deposit formation was associated withcapillary wall thickening, mesangial interposition and expansion,aneurysmal dilitation and intraluminal occlusion of glomerular capillaryloops, and heavy proteinuria.

Given its cross-reactive and distinctive nephritogenic properties, ananalysis of whether anti-DNA-IL/IM formed immune deposits by directinteraction with glomerular Ag was considered. To address thispossibility, the capacity of monoclonal anti-DNA IgG2a Ab, anti-DNAIL/IM to bind to glomerular cell surface antigens was evaluated.Anti-DNA IL/IM produced mesangial, subendothelial and intraluminaldeposits, in vivo after administration to normal mice. By FACS, anti-DNAIL/IM (referred to by hybridoma number H221) bound to mesangial, tubularepithelial and aortic endothelial cell surfaces, whereas surface bindingby isotype-matched anti-DNA antibodies that did not produce glomerularimmune deposits, was not observed. The results are illustrated in FIG.1. (Murine endothelial cells were the kind gift of Dr. Fuad Ziyadeh,Renal Division, University of Pennsylvania.)

Western blots using total cell lysates of component glomerular cellsprobed with anti-DNA antibodies showed that anti-DNA IL/IM reacted withmultiple bands, whereas anti-DNA antibodies that did not form immunedeposits did not. Following biotinylation of cell surface antigen andimmunoprecipitation with anti-DNA IL/IM, a 100 kD band within mesangialcell lysates was identified that was not recognized by an isotypematched control monoclonal anti-DNA antibody.

Thus, anti-DNA IL/IM mAb was demonstrated to bind to murine renalmesangial cells and aortic endothelial cells, and a candidate surfaceprotein target has been identified. This indicates the polyspecificantigen binding properties of this mAb, which is a common feature ofpathogenic SLE antibodies, and may be responsible for binding to cellsurface antigens in the initiation of glomerular immune depositformation.

Sequence analysis of anti-DNA IL/IM (H221) has been carried out. Thecomplete VL-JL sequence (SEQ ID NO:4) and a near complete sequence ofthe VH-DH-JH (SEQ ID NO:6) are shown here.

H221 VL-JL Sequence (SEQ ID NO:4) GAC ATT GTG ATA TCA CAG TCT CCA TCCACC CTG GCT GTG TCA GCA GGA GAG AAG GTC ACT ATG AAC asp ile val ile sergln ser pro ser thr leu ala val ser ala gly glu lys val thr met asn                                CDR I TGC AAA TCC AGT CAG AGT CTG TTCAAC AGT AGA ACC CGA AAG AAC TAC TTG GCT TGG TTC CAG CAG cys lys ser sergln ser leu phe asn ser arg thr arg lys asn tyr leu ala trp phe gln gln                                                    CDR II AAA CCA GGGCAG TCT CCT AAA CTG CTG ATC TAC TGG GCA TCC ACT AGG GAA TCT GGG GTC CCTGAT lys pro gly gln ser pro lys leu leu ile tyr trp ala ser thr arg gluser gly val pro asp CGC TTC ACA GGC AGT GGA TCT GGG ACA GAT TTC ACT CTCACC ATC AGC AGT GTG CAG GCT GAA GAC arg phe thr gly ser gly ser gly thrasp phe thr leu thr ile ser ser val gln ala glu asp                                   CDR III CTG GCA GTT TAT TAC TGC AAGCAA TCT TAT TAT CTT CGG ACG TTC GGT GGA GGC ACC AGG CTG GAA leu ala valtyr tyr cys lys gln ser tyr tyr leu arg thr phe gly gly gly thr arg leuglu H221 VH-DH-JH Sequence (SEQ ID NO:6) GAG GTC CAG CTG CAG CAG CCT GGTGCT GAA CTT GTG AAG TCT GGG GCC TCA GTG AAG CTG glu val gln leu gln glnpro gly ala glu leu val lys ser glu ala ser val lys leu                                               CDR I TCC TGC AAG GCT TCTGAC TTC ACT TTC ACC AGC TAC TGG ATA AAC TGG GTG AAA CAG AGG ser cys lysala ser asp phe thr phe thr ser tyr trp ile asn trp val lys gln arg                                                             CDR II CCTGGA CAA GGC CTT GAG TGG ATT GGA AAA TTT TAT CCT GGT AGT GGT ACT ATT AACTAC pro gly gln gly leu glu trp ile gly lys phe tyr pro gly ser gly thrile asn tyr AGT GAA AAT TTT AAG AAA AAG GCC ACA CTG ACT GTA GAC ACA TCCTCC AGT ACA TCC TAC ser glu asn phe lys lys lys ala thr leu thr val aspthr ser ser ser thr ser tyr ATG CAG CTC AGC AGC CTG ACA TCT GAC GAC TCTGCG GTC TAT TAT TGT GCA AGA GAA CGT met gln leu ser ser leu thr ser aspasp ser ala val tyr tyr cys ala arg glu arg      CDR III CTC CTG GGG TTTGTT TAT TGG GGC CAA GGG ACT CTG GTC ACT GTC TCT ACA GCC AAA ACA leu leugly phe val tyr trp gly gln gly thr leu val thr val ser thr ala lys thrACA GCC CCA TCG GTC TAT CGG GGA TCC TCT AGA GTC GAC CTG CAG GCA TGC AAGCTT GGC ACT thr ala pro ser val tyr arg gly ser ser arg val asp leu glnala cys lys leu gly thr

The results confirm that anti-DNA IL/IM (H221) is a member of the J558VH family and provides reagents to perform the experiments described inthe Research Design and Methods section. Additional sequence analysis isunderway to complete the sequence of the heavy chain. GeneticImmunization against Anti-DNA IL/IM:

Preliminary studies have been performed immunizing AKR×DBA/2 mice withDNA constructs encoding the anti-DNA IL/IM VH or Fv regions and thenchallenging them with a lethal dose of the parent hybridoma cells.Briefly, as shown in FIG. 2A, the VH or Fv regions were cloned into thegenetic immunization vector placing the V regions under control of theCMV promoter. As described in FIG. 2B, the V regions were targetedeither to the cell membrane, to be secreted, to remain in the cytosol,or to be retained in the endoplasmic reticulum using the adenovirus E19protein ER retention signal. Constructs targeted to the lysosomes werealso developed.

Biocca, S. et al. 1990 EMBO J. 9(1):101-108, which is incorporatedherein by reference describes targeting to the cell membrane, secretion,cytosolic localization. Nilsson et al. 1989 Cell 58:707-718, Jackson etal. 1993 J. Cell Biol. 121(2)317-333, and Jackson et al 1990 EMBO J.9:3153-3162 described proteins retained in the endoplasmic reticulumusing the adenovirus E19 protein ER retention signal. Letourneur, F. andR. D. Klausner 1992 Cell 69:1143-1157 describe proteins targeted to thelysosomes.

In preliminary experiments, purified plasmid DNA was inoculated intomice following Bupivacaine pretreatment, and following several booststhe mice were evaluated for antibody responses. Controls included killedhybridoma cells and purified antibody Fv regions. None of theseimmunogens was capable of eliciting a serologic response againstanti-DNA IL/IM Fab fragments (detection was with labeled Staph. proteinG). In preliminary studies, proliferative and cytotoxic T cell responseswere elicited with several of the constructs. The best constructs wereselected for evaluation in a larger group of mice. Groups of fourAKR×DBA/2 mice were immunized once with 100 μg plasmid DNA withBupivacaine simultaneously. One week later, they were sacrificed andevaluated for cytotoxic T cell and proliferative responses.Proliferation was evaluated by stimulating the immune spleenocytes witheither purified H221 mAb (25 or 5=B5 g/well) or with 20,000 or 100,000killed anti-DNA IL/IM hybridoma cells for 72 hours, followed by a pulsewith tritiated thymidine overnight. The results are shown in FIG. 3.

The best proliferative response was induced by the killed hybridomacells. Measurable responses (compared with vector only) were seen formost constructs, but were only modest in magnitude. The proliferativeresponses against purified mAb were significant (>2 standard errorsabove the backbone control) for the mice immunized with killed cells orwith the ER-targeted vaccines. The proliferative responses against thehybridoma cells were significant (>2 standard errors above the backbonecontrol) for both ER-targeted vaccines, and the VH-soluble andVH-cytosolic vaccines. In contrast, the CTL responses elicited werestriking. These results are shown in FIGS. 4A, 4B and 4C.

The immune spleen cells were cultured for 7 days in the presence ofkilled anti-DNA IL/IM stimulators (20:1 ratio of spleen cells tostimulators), in the presence of concanavalin A for the first 2 days,then with stimulators only. The anti-DNA IL/IM hybridoma cells werelabeled with 51Cr, and lysis determined in round bottomed microtiterplates. The mean=B1 standard deviation % specific lysis (as noted abovefor CD4) is shown for various effector:target ratios. Interestingly, allof the constructs except one (soluble heavy chain V region or VH sol)elicited CTL activity as good or better than killed cells, which was thepositive control for the assay. In particular, exceptional responseswere seen for the cytosolic and ER targeted constructs. This indicatesthat targeting to these compartments boosts the CTL responses seen.

These studies indicate good to excellent induction of CTL responses bymost of the DNA vaccines evaluated. This experiment can serve as anexample of the strategy used to select particular vaccines to carry onin the study while excluding other. Thus, the Fv construct targeted tothe cytosol elicited a CTL response that was clearly higher (>2 standarderrors) than the other constructs. Based on this analysis, this vaccineinvites further evaluation. Similarly, the VH soluble vaccine did notelicit a good CTL response. Therefore, this vaccine would be eliminatedfrom further analysis, as it would not be expected to elicit protectiveresponses based on the hypotheses generated in the CD4 system.

An additional experiment with this vaccine revealed that this was indeedthe case. In this experiment, groups of 5 or 6 mice were immunized 3times with 100=B5 g's of the DNA vaccines. They were then challengedwith anti-DNA IL/IM hybridoma cells intraperitoneally. Four weeks later,when tumors developed in the control animals, all of the mice weresacrificed and evaluated for tumor burden and amount of ascites. Theproportion of mice developing tumors was also quantified. The resultsare shown in FIGS. 5A, 5B and 5C.

FIG. 5A shows the proportion of mice in which tumors developed. FIG. 5Bshows the mean tumor mass in the groups, as well as the values forindividual mice. FIG. 5C similarly shows the volume of malignant ascitesobtained. Note that in this particular experiment, mice immunized withkilled cells (our positive control) did not differ very much from miceimmunized with vector only (our negative control) by any measure. Inspite of this, several of the DNA vaccines showed clear protectiveability, particularly the FV cytosolic targeted vaccine and both the FVand VH endoplasmic reticulum (ER) targeted vaccines. This correlatesfairly well with these vaccines' ability to elicit CTL responses. The VHsoluble vaccine showed no protective activity, which correlates with itsfailure to show CTL activity.

Example 2

Several human diseases are associated with pathologic proliferation of Band T cells. This includes malignancies or hyperproliferative diseasessuch as lymphoma and leukemia, and autoimmune diseases, such as SystemicLupus Erythematosus (SLE) where pathogenic autoantibodies mediate tissueinjury. Current therapy for these diseases is inadequate and treatmentis associated with a high incidence of side-effects. A more logicalapproach to therapy for such diseases is to specifically eliminate thepathogenic cells. This might be accomplished by active immunotherapytargeting their variable regions. Active immunization against V regionshas the potential of eliminating the pathogenic cells. Furthermore, byeliciting protective immunity, reemergence of pathogenic clones can beeliminated. However, the immune response elicited by immunization alsohas the potential to produce detrimental consequences. For example,ideally vaccination against autoantibody producing B cell V regionsshould elicit cytotoxic T cell (CTL) responses, deleting the pathogenicB cells, limiting autoantibody production. If however helper T cells,(i.e. TH₂ type responses) were elicited, autoantibody production mightin fact be increased to the detriment of the patient. Therefore, activeimmunization against pathogenic B or T cell V regions should be designedto elicit desired immune responses (such as CTL responses), whilelimiting potentially detrimental responses (such as TH₂ responses).

DNA vaccination against an autoantibody V region has been evaluated asfollows. The autoantibody-producing hybridoma anti-DNA IL/IM (alsocalled H221) was selected from a large panel of hybridomas derived fromMRL-1pr/lpr mice; when anti-DNA IL/IM producing hybridoma cells areadministered intraperitoneally to histocompatible mice, is they produceglomerulonephritis, characterized by dense intramembranous andintraluminal deposits, associated with capillary wall thickening,mesangial interposition and expansion, aneurysmal dilation andintraluminal occlusion of glomerular capillary loops, and heavyproteinuria. This provided an in vivo system in which the efficacy ofidiotypic DNA vaccination targeting a pathogenic autoantibody V regionin eliciting protective immunity was evaluated. To investigate thepossibility of enhancing the immunogenicity of these DNA vaccines, geneexpression was targeted to specific intracellular versus extracellularcompartments (cytosolic, endoplasmic reticulum (ER) for secretion and ERfor retention). Vaccination against a single V region (the V_(H) region)and against the entire Fv (V_(H) linked to V_(L)) fragment wereemployed. The results indicate that DNA inoculation against the H221V_(H) and Fv regions elicits specific cellular immune responses,particularly potent CTL responses, with enhancement in CTL activity bytargeting the V region to be expressed in the cytosol or to be retainedin the ER. Furthermore, idiotypic DNA vaccination elicited protectiveimmunity against H221 cells, particularly when the gene product wastargeted for retention in the ER.

Materials and Methods

DNA Constructs

pBBkan is a eukaryotic expression vector which utilizes a CMV promoterand an RSV enhancer to direct transcription (FIG. 8). This was initiallymodified by subcloning a secretory leader from mouse IgG or acytoplasmic leader into the multiple cloning site between the NotI andXbaI sites. The vector with the secretory leader was the parent vectorfor the secreted and the ER-retained vaccines. Likewise, the vector withthe cytoplasmic leader was the parent vector for the cytoplasmicallyexpressed vaccines. The H221 V_(H) and V_(L) regions were amplified bythe polymerase chain reaction (PCR) and recombinant PCR was used togenerate the Fv coding sequences (Srikatan, et al. 1994 AIDS 8:1525-32,which is incorporated herein by reference). An XbaI restriction site wascloned into the 5′ end of the single heavy chain products and the 5′ endof the kappa light chain product (Fv). The CPR products were restrictiondigested and then purified from 2% low-melting agarose by standardphenol-chloroform extraction, gel purified and either ligated directlyinto the vector (as with the leader sequences) or ligated to otherfragments and used as templates for further PCR reactions splicing onthe 3′ targeting sequences (CD4 TM to E19, Fv or Vh to CD4-E19) whereindicated. The E19 signal sequence was amplified from a CD8-E19(Nilsson, et al. 1989 CELL 58:707-718, which is incorporated herein byreference). The primers used are listed in Table 3. The amino acidsequences for the Immunoglobulin Leader (ER Targeting for Secretion orRetention) (SEQ ID NO:22), Cytosolic Leader (SEQ ID NO:23), H221 VLRegion (SEQ ID NO:24), Linker Peptide (SEQ ID NO:25), H221 VH Region(SEQ ID NO:26) and CD4 transmembrane and E19 Cytoplasmic Domains (for ERRetention) (SEQ ID NO:27) are listed in Table 4. The ImmunoglobulinLeader (SEQ ID NO:22) was a murine immunoglobulin leader that was usedto target the gene product to the ER for secretion or ER retention. TheCytosolic Leader (SEQ ID NO:23) was a sequence previously reported forintracellular expression of antibodies (Biocca, et al. 1990 EMBO J.9:101-108, which is incorporated herein by reference). The VH and VLsequences (SEQ ID NO:24 and SEQ ID NO:26, respectively) were determinedfollowing cloning of the PCR products. The Linker Peptide (SEQ ID NO:25)was used for functional expression of Fv regions. The human CD4transmembrane region was combined with the adenovirus E19 Targetingsequence for ER retention (SEQ ID NO:27).

These PCR products were cloned into pBBkan and transformed into DH5alpha E. coli (Life Technologies Inc., Gaithersburg, Md.) and cloneswith correct restriction patterns were sequenced with the ABIfluorescent sequencing kit (Applied Biosystems Inc., Foster City,Calif.). Products were purified and dried as per kit instructions andgel running and analysis were performed by the University ofPennsylvania Cancer Center Core sequencing Facility. Sequence analysisreveals that H221 utilizes the J558 V_(H) and J^(H)3 genes, paired withthe VK1 genes. The vector, constructs, and insert sequences are shown inFIG. 8 and Table 4. Plasmid preparations were grown in Super Broth (1.2%w/v Difco tryptone, 2.4% w/v Difco yeast extract, 0.5% v/v glycerol, 72mM potassium phosphate dibasic, 28 mM potassium phosphate monobasic) andpurified using Qiagen 500 tips according to the manufacturer's protocol(Qiagen Inc., Chatsworth, Calif.). DNA inoculation was carried out withbupivacaine pre-treatment (Wang, et al. 1993 Proc. Natl. Acad. Sci. USA90:4156-4160, which is incorporated herein by reference).

Proliferation Assay

Individual spleens from the vaccinated mice are extracted in a sterilefashion, gently disrupted in RPMI and treated with Gey's solution tolyse the red blood cells. Splenocytes are plated in 96-well flat-bottomtissue culture plates (Falcon 3072; Becton Dickinson, Franklin Lakes,N.J.) at 500,000 per well in a 200 μL final volume. Triplicate wells areexposed to media alone, 2 μg/mL concanavalin A, 5 and 25 μg/mL Mab H221,and 5:1 or 25:1 (splenocyte: stimulator) mitomycin C killed H221hybridoma cells. 10⁶ hybridoma cells/mL were treated with 25 μg/mLmitomycin C for 45 minutes at 37° C. in PBS then washed three times withPBS to remove toxin. After three days of culture at 37° C. with 5% CO₂,1 μCi tritiated thymidine (NEN Life Sciences, Boston Mass.) is added in20 μL media and 18 hours later the plates are harvested on the TomtecHarvester 96 (Tomtec, Orange, Conn.). Filtermats are dried and thencounted with Beta scint on the Microbeta 1450 scintillation counter(Wallac Inc., Gaithersburg, Md.).

Cytotoxic T Cell Assay

Splenocytes are cultured at 5×10⁵/mL with 2 μg/mL concanavalin A andmitomycin C treated stimulators at 20:1 splenocyte to stimulator ratiofor 24 hours at which point the media is changed to remove the mitogen.After an additional four days of culture the cells are collected into 10mL of media and centrifuged over Ficoll for 20 minutes at 1500 g. Cellswere then counted and plated at 100:1, 50:1, 25:1 and 12.5:1 effector:target with a constant number of target cells in each round bottomedwell. The target cells are prepared by resuspending 10⁷ viable H221cells in 250 μL media and adding 75 μCi ⁵¹Cr-sodium chromate (NEN/LifeSciences Inc., Boston, Mass.) and incubating for 2 hours at roomtemperature. These cells were then washed 3 times with RPMI completemedia and resuspended for plating. Plates were centrifuged at 800 rpmfor 2 minutes and then incubated at 37° C. for 5 and 16 hours at whichtimes 50 μL of the supernatant was carefully removed and counted withOptiphase scintillation fluid (Wallace Inc., Gaithersburg, Md.) in thescintillation counter. Percent specific lysis was calculated as per thefollowing equation:

% specific lysis=Experimental Release−Spontaneous Release/TotalRelease−Spontaneous Release

(Spontaneous release are those cpm released from cells incubated inmedia alone and total release are those released by total lysis oftarget cells by incubation in 1% SDS.)

In Vivo Tumor Challenge

Mice were primed with pristane (1 mL intraperitoneally) at the time ofthe last of 3 DNA inoculations. One week later the mice (6 per group)were injected intraperitoneally with 10⁶ viable H221 hybridoma cellswhich had been washed twice and resuspended in sterile-PBS at 10⁷/mL.Mice were checked weekly for the first two weeks and then twice weeklythereafter for signs of tumor growth (palpable masses and/or ascites).At 4 weeks following challenge, the mice were sacrificed, solid tumorswere dissected and weighed to quantify tumor mass. In mice withmalignant ascites, the cellular content of ascites was also weighed andadded to the tumor mass.

Anti-DNA Antibody Assay

Sera from mice were tested in an anti-dsDNA ELISA (Madaio, et al. 1984J. Immunol. 132:872, which is incorporated herein by reference).

Results

Idiotypic DNA Inoculation Elicits Specific Immune Responses

In preliminary experiments, purified plasmid DNA was inoculated intoAKR×DBA/2 mice, and following several boosts the mice were evaluated forcellular and humoral immune responses. Controls included killedhybridoma cells and purified H221 mAb. Both lymphocyte proliferative andcytotoxic T cell responses were elicited against H221 cells with theconstructs in the absence of a detectable serologic response againstanti-DNA IL/IM Fab fragments (detection was with labeled Streptococcalprotein G). The cellular immune responses were evaluated more closely.Groups of four AKR×DBA/2 mice were inoculated once with plasmid DNA withbupivacaine-HCl simultaneously. One week later, they were sacrificed andevaluated for cytotoxic T cell and proliferative responses. The resultsare shown in FIGS. 9 and 10.

In the experiments to evaluate the proliferative response ofspleenocytes following a single DNA inoculation, mice were inoculatedonce with 100 μg plasmid DNA in 0.25% Bupivacaine, and one week later,the spleenocytes were removed and incubated either in media alone, withkilled H221 cells or with H221 Fab. Proliferation was evaluated bystimulating the immune spleenocytes with either purified H221 mAb (25 or5 μg/well) or with killed anti-DNA IL/IM hybridoma cells (20,000 or100,000/well). The positive control was inoculation with killed H221cells (killed cells), with the negative control inoculation with vectoronly. Following a 3 day culture, the cells were pulsed overnight withtritiated thymidine, and counts per minute (CPM) incorporateddetermined. The mean ΔCPM (experimental minus media only)±the standarddeviation of triplicate wells is shown for two concentrations of Fab andtwo concentrations of killed H221 cells. The results are shown in FIG.9.

In the experiments to evaluate the cytotoxic T cell (CTL) responsefollowing a single DNA inoculation, mice were inoculated one with 100 μgplasmid DNA in 0.25% Bupivacaine, and one week later, the spleenocyteswere removed and incubated with concanavalin A and killed H221 cells(20:1 effector:stimulator ration). After 24-48 hours the Con A wasremoved, the cells fed, and the culture continued for 5 days total. Thepositive control was inoculation with killed H221 cells (killed cells),with the negative control inoculation with vector only. Following the 5day culture, the effector cells were isolated by discontinuous gradientcentrifugation, and used to lyse⁵¹Cr labeled target cells at the variousrations shown. % specific lysis was calculated as noted in Materials andMethods. The mean ± standard deviation % specific lysis is shown fortriplicate determinations. The controls (killed H221 cells and vectoronly) are shown in all three graphs for reference. The results are shownin FIGS. 10A, 10B and 10C.

As in other experiments, the best proliferative responses were inducedby the killed hybridoma cells (FIG. 9). Measurable responses (comparedwith vector only) were seen for most constructs, but were only modest inmagnitude. The proliferative responses against purified mAb weresignificant (>2 standard errors above the backbone control) for the miceimmunized with killed cells or with the ER-retained vaccines. Theproliferative responses against the hybridoma cells were significant (>2standard errors above the backbone control) for both ER-retainedvaccines, and the V_(H)-soluble and V_(H) cytosolic vaccines.

In contrast, the CTL responses elicited were striking (FIG. 10). All ofthe constructs except the soluble heavy chain V region (V_(H) sol)elicited CTL activity as good or better than killed cells, which was thepositive control for the assay. In other experiments, V_(H) sol elicitedsignificant CTL responses compared with controls (22% compared with 10%specific lysis at 100:1 effector: target ratio for the V_(H) solconstruct versus control in a typical experiment). Exceptional responseswere seen for the cytosolic and ER targeted constructs indicating thattargeting to these compartments boots the CTL responses. The Fvconstructs generally elicited more potent CTLs, with the exception ofthe ER-retained vaccines.

Protection from Hybridoma Challenge

To evaluate the efficacy of these DNA vaccines in protecting fromchallenge with autoantibody-producing hybridoma cells in vivo, groups of5-6 mice were immunized three times at biweekly intervals with 100 μg ofthe DNA vaccines in bupivacaine. The mice were challenged with liveanti-DNA LK/IM hybridoma cells (H221 cells) intraperitoneally. Fourweeks letter, the mice were evaluated for tumor burden and ascites. Allmice with tumors or ascites were sacrificed, tumors excised and Tumorburden determined. The results are shown in FIGS. 11A and 11B. FIG. 11Ashows the tumor mass for each vaccinated group. The mean value is shownin a bar graph, with the values for individual mice superimposed. FIG.11B shows the proportion of mice at 4 weeks developing tumors withineach group is shown. Several of the DNA vaccines showed specificeffects, as evidenced by reduced mean tumor burden. Both the F_(V) ANDV_(H) ER retained vaccines prevented tumor formation in 6/6 and 4/5miche respectively, compared with 3/6 immunized with killed cells and2/5 receiving the vector control.

Nearly all of the mice evaluated demonstrated an increase in serumanti-DNA titers compared with baseline responses, although this wasrelatively small. There were no marked differences between groups inthis parameter, although lower anti-DNA levels were seen in the groupimmunized with the Fv ER-retained vaccine compared with the vector onlycontrols (a titer increase of 0.83±0.41 for the Fv ER-retained vaccinescompared with 1.6±0.55 in the vector control mice, p=0.025 Student'st-Test). Thus, the most potent vaccine did significantly lowercirculating anti-DNA levels in this model as well.

Immune Responses in Survivors

CTL activity and proliferation in response to killed H221 cells inseveral mice that survived tumor challenge were evaluated. This included3 mice immunized with killed cells, 3 mice immunized with the cytosolicFv construct, and 3 mice immunized with the ER-retained Fv construct.The mice were sacrificed at week 5 (38 days following the initial tumorchallenge) and proliferation of spleenocytes in response to killed H221cells determined as described in FIG. 9. The mean CPM incorporated oftriplicate wells is shown for individual mice, labeled according to thevaccine they received. The CTL responses had waned (the mice had notbeen boosted prior to the assay) with the highest response (13% specificlysis at a 50:1 effector:target ratio) seen in the mice immunized withthe ER retained construct, but with similar responses seen in the other(7% in the killed cells immunized and 7% in the Fv cytosolic constructimmunized mice). In contrast, the proliferative responses were stillvigorous (FIG. 12). Mice initially immunized with killed cells or thecytosolic targeted Fv construct responded to killed H221 cells in vitro(mean values of 7,488±5,864 CPM dn 6,663±4,171 CPM respectively,compared with 721±409 and 5,044±1902 for media alone controls). Incontrast, those mice initially immunized with the ER retained Fvconstruct responded to killed H221 cells in vitro (mean 37,351±9,004 CPMversus 4,651±1,345 with media alone). This exuberant proliferativeresponse was mirrored by enhanced IL-2 production in parallel cultures(on the average, there was twice as high in the Fv ER-retained vaccinescompared with those immunized with killed cells). Together, theseresults indicate a strong cellular proliferative response in the miceimmunized with the Fv ER-retained vaccine who survived challenge.

Discussion

These studies indicate that idiotypic DNA vaccination is capable ofeliciting a cellular immune response in the absence of a detectablehumoral immune response. Furthermore, for most of the vaccines evaluatedthe CTL response was equivalent to or surpassed the CTL responseelicited by killed cells (FIG. 10). In contrast to the killed cellvaccine, however, the proliferative response (which typically correlateswith T_(H) responses) was barely detectable following DNA vaccination(FIG. 9). Thus, it is possible with idiotypic DNA vaccination to elicita potent CTL response with only a slight T_(H) response. This allowsinvestigation of the efficacy of CTL's in inducing protective immunity.The immunogenicity of these idiotypic DNA vaccines was markedly enhancedby targeting the DNA vaccine to specific intracellular compartments,particularly with regard to CTL responses (FIG. 10). Targeting to thecytosol and retention in the ER enhanced the CTL responses, as would beexpected for MHC class I restricted responses. The Fv vaccines (V_(H)and V_(L) regions together) were observed to be more immunogenic thanthe V_(H) region alone. This is likely due to the greater number ofepitopes presented to the immune system. A direct effect of the linkerpeptide can not be ruled out.

Idiotypic DNA vaccination is capable of protecting mice fromhistocompatible tumor challenge if the vaccine is targeted to theappropriate intracellular compartment (FIG. 11). This protectiveresponse is clearly due to cellular immunity, as humoral immunity wasnot detectable in this system even in survivors from tumor challenge.The protection seen most likely was due to the CTL responses elicited,as a potent CTL response was elicited by the ER-retained vaccines, whichwere the “most protective” vaccines. This suggests that concentration ofthe antigen in the ER and elicitation of CTL's results in protectiveimmunity. Other factors could be contributing to the protectiveresponses such as the presence of non-self antigenic determinants in theconstructs used for vaccination. The mice immunized with the ER-retainedvaccine who survived tumor challenge mounted an impressive proliferativeresponse (FIG. 12). This indicates that the ER-retained Fv DNA vaccineprimed the immune system for a secondary response to the tumor challengewith marked expansion of idiotype-specific T cells. This proliferativeresponse was not as high in the survivors who were initially immunizedwith the killed cells or the Fv cytosolic vaccine. This may relate tothe timing of the immune response following challenge, as only a singletime point was sampled in this study. However, a selective effect of theER-retained vaccine in priming for a potent secondary response issupported by the complete protection from tumor challenge in this group.

The ability to manipulate the expression, cellular localization, andother parameters of DNA vaccines renders them particularly suited forinvestigations into the nature of induced immune responses which canlead to protection in vivo. DNA-vaccination has been used against avariety of model tumor antigens, including our studies using murinelymphoma cells expressing human CD4, a similar system using thebeta-galactosidase gene, human carcinoembryonic antigen, a singleepitope from a mutant form of the human p53 gene. These studiesestablish the utility of DNA vaccination against model tumor antigens.All of the antigens used were in fact foreign proteins which typicallyare much more immunogenic than tumor self-antigens which would beencountered clinically.

The use of the V_(H) and V_(L) regions of a murine lymphoma in anexpression vector also encoding the human Cγ1 and C_(κ) with or withoutlinked expression of human GM-CSF has been reported (Syrengelas, et al.1996 Nature Medicine 2:1038-1041, which is incorporated herein byreference). The immunogen thus had foreign antigenic determinants linkedto the self-V regions of interest. Intramuscular or intradermal DNAinoculation with these constructs resulted in an anti-idiotypic antibodyresponse, as well as partial protection from tumor challenge in vivo.The linked expression of human GM-CSF markedly enhanced the responseselicited. DNA immunization induced immune responses against a weak,otherwise unrecognized tumor antigen, this was dependent on additionalstimuli with the DNA (i.e the human constant regions and GM-CSF).

Isolated syngeneic V region DNA immunization has been shown to becapable of eliciting protective immune responses in a murine model ofautoimmune disease. DNA based immunization against the murine Vβ8.2 genehas been shown to protect H-2^(u) mice from experimental autoimmuneencephalomyelitis (EAE) (Waisman, et al. 1996 Nature Medicine 2:899-905,which is incorporated herein by reference). Cellular immune responseswere demonstrated, and that data suggested that the DNA immunization hadshut off the pathogenic T cells, which are dominated by clonesexpressing Vβ8.2 in this system. No evidence of deletion ofVβ8.2-bearing cells with their vaccination approach (which used anisolated V region without associated a leader peptide or the CDR3) wasshown.

TABLE 1 Picornavirus Family Genera: Rhinoviruses: (Medical) responsiblefor ˜50% cases of the common cold. Etheroviruses: (Medical) includespolioviruses, coxsackieviruses, echoviruses, and human enterovirusessuch as hepatitis A virus. Apthoviruses: (Veterinary) these are the footand mouth disease viruses. Target antigens: VP1, VP2, VP3, VP4, VPGCalcivirus Family Genera: Norwalk Group of Viruses: (Medical) theseviruses are an important causative agent of epidemic gastroenteritis.Togavirus Family Genera: Alphaviruses: (Medical and Veterinary) examplesinclude Senilis viruses, RossRiver virus and Eastern & Western Equineencephalitis. Reovirus: (Medical) Rubella virus. Flariviridue FamilyExamples include: (Medical) dengue, yellow fever, Japanese encephalitis,St. Louis encephalitis and tick borne encephalitis viruses. Hepatitis CVirus: (Medical) these viruses are not placed in a family yet but arebelieved to be either a togavirus or a flavivirus. Most similarity iswith togavirus family. Coronavirus Family: (Medical and Veterinary)Infectious bronchitis virus (poultry) Porcine transmissiblegastroenteric virus (pig) Porcine hemagglutinating encephalomyelitisvirus (pig) Feline infectious peritonitis virus (cats) Feline entericcoronavirus (cat) Canine coronavirus (dog) The human respiratorycoronaviruses cause ˜40 cases of common cold. EX. 224E, 0C43 Note -coronaviruses may cause non-A, B or C hepatitis Target antigens: E1 -also called M or matrix protein E2 - also called S or Spike protein E3 -also called HE or hemagglutin-elterose glycoprotein (not present in allcoronaviruses) N - nucleocapsid Rhabdovirus Family Genera: VesiliovirusLyssavirus: (medical and veterinary) rabies Target antigen: G protein Nprotein Filoviridue Family: (Medical) Hemorrhagic fever viruses such asMarburg and Ebola virus Paramyxovirus Family: Genera: Paramyxovirus:(Medical and Veterinary) Mumps virus, New Castle disease virus(important pathogen in chickens) Morbillivirus: (Medical and Veterinary)Measles, canine distemper Pneuminvirus: (Medical and Veterinary)Respiratory syncytial virus Orthomyxovirus Family (Medical) TheInfluenza virus Bungavirus Family Genera: Bungavirus: (Medical)California encephalitis, LA Crosse Phlebovirus: (Medical) Rift ValleyFever Hantavirus: Puremala is a hemahagin fever virus Nairvirus(Veterinary) Nairobi sheep disease Also many unassigned bungavirusesArenavirus Family (Medical) LCM, Lassa fever virus Reovirus FamilyGenera: Reovirus: a possible human pathogen Rotavirus: acutegastroenteritis in children Orbiviruses: (Medical and Veterinary)Colorado Tick fever, Lebombo (humans) equine encephalosis, blue tongueRetrovirus Family Sub-Family: Oncorivirinal: (Veterinary) (Medical)feline leukemia virus, HTLVI and HTLVII Lentivirinal: (Medical andVeterinary) HIV, feline immunodeficiency virus, equine infections,anemia virus Spumavirinal Papovavirus Family Sub-Family: Polyomaviruses:(Medical) BKU and JCU viruses Sub-Family: Papillomavirus: (Medical) manyviral types associated with cancers or malignant progression ofpapilloma Adenovirus (Medical) EX AD7, ARD., O.B. - cause respiratorydisease - some adenoviruses such as 275 cause enteritis ParvovirusFamily (Veterinary) Feline parvovirus: causes feline enteritis Felinepanleucopeniavirus Canine parvovirus Porcine parvovirus HerpesvirusFamily Sub-Family: alphaherpesviridue Genera: Simplexvirus (Medical)HSVI, HSVII Varicellovirus: (Medical - Veterinary) pseudorabies -varicella zoster Sub-Family betaherpesviridue Genera: Cytomegalovirus(Medical) HCMV Muromegalovirus Sub-Family: Gammaherpesviridue Genera:Lymphocryptovirus (Medical) EBV - (Burkitts lympho) RhadinovirusPoxvirus Family Sub-Family: Chordopoxviridue (Medical - Veterinary)Genera: Variola (Smallpox) Vaccinia (Cowpox) Parapoxivirus - VeterinaryAuipoxvirus - Veterinary Capripoxvirus Leporipoxvirus SuipoxvirusSub-Family: Entemopoxviridue Hepadnavirus Family Hepatitis B virusUnclassified Hepatitis delta virus

TABLE 2 Bacterial pathogens Pathogenic gram-positive cocci include:pneumococcal; staphylococcal; and streptococcal. Pathogenicgram-negative cocci include: meningococcal; and gonococcal. Pathogenicenteric gram-negative bacilli include: enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigellosis;hemophilus; chancroid; brucellosis; tularemia; yersinia (pasteurella);streptobacillus moniliformis and spirillum; listeria monocytogenes;erysipelothrix rhusiopathiae; diphtheria; cholera; anthrax; donovanosis(granuloma inguinale); and bartonellosis. Pathogenic anaerobic bacteriainclude: tetanus; botulism; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include: syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude: actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include rickettsial and rickettsioses. Examplesof mycoplasma and chlamydial infections include: mycoplasma pneumoniae;lymphogranuloma venereum; psittacosis; and perinatal chlamydialinfections. Pathogenic eukaryotes Pathogenic protozoans and helminthsand infections thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; pneumocystis carinii; babesiosis;giardiasis; trichinosis; filariasis; schistosomiasis; nematodes;trematodes or flukes; and cestode (tapeworm) infections.

TABLE 3 Primers used for PCR and recombinant PCR Secretory andER-retained Leaders: 5′ GGGCGGCCGC AATGGACATG AGGGTCCCCG CTCAGCTCCTGGGGCTCCTG (SEQ ID NO:8) 3′ CCTCTAGAAC ATTTGGCACC TGGGAGCCAG AGCAGCAGGAGCCCCAGGAG C (SEQ ID NO:9) Cytoplasmic Leader: 5′ GGGCGGCCGC AATGGGATGGAGCTGTAAGA GGCGCTCCTC GGAAG (SEQ ID NO:10) 3′ CCCTCTAGAG TGGACACCAGCTGTAGCTGT TTCTTCCGAG GAGCG (SEQ ID NO:11) CD4 Transmembrane: 5′GTGCAGCCCA TGGCCCTGAT TGTG (SEQ ID NO:12) 3′ TTCATTGGGC TAGGCATCTTCTTCAGATCT AGGTGC (SEQ ID NO:13) ER-retention signal (adenovirus E19):5′ TTCTTCAGAT CTAGGCGCAG TTTTATTGAT GAA (SEQ ID NO:14) 3′ CGTAAAACGCGTTTAAGGCA TTTCTTTTC (SEQ ID NO:15) 5′ Vkappa for Fv expression:GGGGTTCTAG AGACATTGTG ATATCMCARW CTC (SEQ ID NO:16) 3′ CL Primer(Written antiparallel) for expression with linker peptide: CTGATAAGATTTAGATTCGG AGCCAGAACC GGAAGATTTA CCTTCTGCAG CATCAGCCCG (SEQ ID NO:17) 5′VH2 primer for single chain expression: GGGGTTCTAG AGAGGTCCAG CTGCARCARYCTGG (SEQ ID NO:18) 5′ VH Primer for expression with linker peptide:GGCTCCGAAT CTAAATCTTA TCAGGAGGTC CAGCTGCARC ARYCTGG (SEQ ID NO:19) 3′IgG2a CH1 for transmembrane/ER retention: ATAGACCATG GGGGCTGTTG TTTTGGC(SEQ ID NO:20) 3′ IgG2a CH1 for soluble secretion: ATAGAACGCG TGTCAGGCTGTTGTTTTGGC (SEQ ID NO:21) M = A or C R = A or G W = T or A Y = T or C

TABLE 4 Immunoglobulin Leader (ER Targeting for Secretion or Retention):Met asp met arg val pro ala gln leu leu gly leu leu leu leu trp (SEQ IDNO:22) leu pro gly ala lys cys ser arg Cytosolic Leader: Met gly trp sercys lys arg arg ser ser glu glu thr ala thr ala (SEQ ID NO:23) gly valhis ser arg H221 VL Region: Asp ile val ile ser gln ser pro ser thr leuala val ser ala gly (SEQ ID NO:24) glu lys val thr met asn cys lys serser gln ser leu phe asn ser arg thr arg lys asn tyr leu ala trp phe glngln lys pro gly gln ser pro lys leu leu ile tyr trp ala ser thr arg gluser gly val pro asp arg phe thr gly ser gly ser gly thr asp phe thr leuthr ile ser ser val gln ala glu asp leu ala val tyr tyr cys lys gln sertyr tyr leu arg thr phe gly gly gly thr arg leu glu Linder Peptide: Argala asp ala ala glu gly lys ser ser gly ser gly ser glu ser (SEQ IDNO:25) lys ser tyr gln gly ser glu ser lys ser tyr gln H221 VH Region:Glu val gln leu gln gln ser gly ala glu leu val lys ser gly ala (SEQ IDNO:26) ser val lys leu ser cys lys ala ser gly phe thr phe thr ser tyrtrp ile asn trp val lys gln arg ala gly gln gly leu glu trp ile gly asnile tyr pro gly ser asn thr ile asn tyr ser glu asn phe lys lys lys alathr leu thr val asp thr ser ser ser thr ala tyr met gln leu ser ser leuthr ser asp asp ser ala val tyr tyr cys ala arg glu arg leu leu gly pheval tyr trp gly gln gly thr leu val thr val ser thr ala lys thr thr alaCD4 transmembrane and E19 cytoplasmic Domains (for ER retention): Metala leu ile val leu gly gly val ala gly leu leu leu phe ile (SEQ IDNO:27) gly leu gly ile phe phe arg ser arg arg ser phe ile asp glu lyslys met pro

27 6 amino acids amino acid both peptide unknown 1 Asp Lys Gln Thr LeuLeu 1 5 4 amino acids amino acid both peptide unknown 2 Lys Asp Glu Leu1 6 amino acids amino acid both peptide unknown 3 Asp Glu Lys Lys MetPro 1 5 330 base pairs nucleic acid both both cDNA unknown CDS 1..330 4GAC ATT GTG ATA TCA CAG TCT CCA TCC ACC CTG GCT GTG TCA GCA GGA 48 AspIle Val Ile Ser Gln Ser Pro Ser Thr Leu Ala Val Ser Ala Gly 1 5 10 15GAG AAG GTC ACT ATG AAC TGC AAA TCC AGT CAG AGT CTG TTC AAC AGT 96 GluLys Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30 AGAACC CGA AAG AAC TAC TTG GCT TGG TTC CAG CAG AAA CCA GGG CAG 144 Arg ThrArg Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln 35 40 45 TCT CCTAAA CTG CTG ATC TAC TGG GCA TCC ACT AGG GAA TCT GGG GTC 192 Ser Pro LysLeu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 CCT GAT CGCTTC ACA GGC AGT GGA TCT GGG ACA GAT TTC ACT CTC ACC 240 Pro Asp Arg PheThr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 ATC AGC AGTGTG CAG GCT GAA GAC CTG GCA GTT TAT TAC TGC AAG CAA 288 Ile Ser Ser ValGln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 TCT TAT TAT CTTCGG ACG TTC GGT GGA GGC ACC AGG CTG GAA 330 Ser Tyr Tyr Leu Arg Thr PheGly Gly Gly Thr Arg Leu Glu 100 105 110 110 amino acids amino acid bothpeptide unknown 5 Asp Ile Val Ile Ser Gln Ser Pro Ser Thr Leu Ala ValSer Ala Gly 1 5 10 15 Glu Lys Val Thr Met Asn Cys Lys Ser Ser Gln SerLeu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Phe Gln GlnLys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr ArgGlu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala ValTyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Tyr Leu Arg Thr Phe Gly Gly Gly ThrArg Leu Glu 100 105 110 423 base pairs nucleic acid both both cDNAunknown CDS 1..427 6 GAG GTC CAG CTG CAG CAG CCT GGT GCT GAA CTT GTG AAGTCT GGG GCC 48 Glu Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys SerGly Ala 1 5 10 15 TCA GTG AAG CTG TCC TGC AAG GCT TCT GAC TTC ACT TTCACC AGC TAC 96 Ser Val Lys Leu Ser Cys Lys Ala Ser Asp Phe Thr Phe ThrSer Tyr 20 25 30 TGG ATA AAC TGG GTG AAA CAG AGG CCT GGA CAA GGC CTT GAGTGG ATT 144 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu TrpIle 35 40 45 GGA AAA TTT TAT CCT GGT AGT GGT ACT ATT AAC TAC AGT GAA AATTTT 192 Gly Lys Phe Tyr Pro Gly Ser Gly Thr Ile Asn Tyr Ser Glu Asn Phe50 55 60 AAG AAA AAG GCC ACA CTG ACT GTA GAC ACA TCC TCC AGT ACA TCC TAC240 Lys Lys Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ser Tyr 6570 75 80 ATG CAG CTC AGC AGC CTG ACA TCT GAC GAC TCT GCG GTC TAT TAT TGT288 Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 8590 95 GCA AGA GAA CGT CTC CTG GGG TTT GTT TAT TGG GGC CAA GGG ACT CTG336 Ala Arg Glu Arg Leu Leu Gly Phe Val Tyr Trp Gly Gln Gly Thr Leu 100105 110 GTC ACT GTC TCT ACA GCC AAA ACA ACA GCC CCA TCG GTC TAT CGG GGA384 Val Thr Val Ser Thr Ala Lys Thr Thr Ala Pro Ser Val Tyr Arg Gly 115120 125 TCC TCT AGA GTC GAC CTG CAG GCA TGC AAG CTT GGC ACT 423 Ser SerArg Val Asp Leu Gln Ala Cys Lys Leu Gly Thr 130 135 140 141 amino acidsamino acid both peptide unknown 7 Glu Val Gln Leu Gln Gln Pro Gly AlaGlu Leu Val Lys Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys AlaSer Asp Phe Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln ArgPro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Lys Phe Tyr Pro Gly Ser GlyThr Ile Asn Tyr Ser Glu Asn Phe 50 55 60 Lys Lys Lys Ala Thr Leu Thr ValAsp Thr Ser Ser Ser Thr Ser Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu ThrSer Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Leu Leu GlyPhe Val Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Thr AlaLys Thr Thr Ala Pro Ser Val Tyr Arg Gly 115 120 125 Ser Ser Arg Val AspLeu Gln Ala Cys Lys Leu Gly Thr 130 135 140 50 base pairs nucleic acidsingle linear DNA unknown 8 GGGCGGCCGC AATGGACATG AGGGTCCCCG CTCAGCTCCTGGGGCTCCTG 50 51 base pairs nucleic acid single linear DNA unknown 9CCTCTAGAAC ATTTGGCACC TGGGAGCCAG AGCAGCAGGA GCCCCAGGAG C 51 45 basepairs nucleic acid single linear DNA unknown 10 GGGCGGCCGC AATGGGATGGAGCTGTAAGA GGCGCTCCTC GGAAG 45 45 base pairs nucleic acid single linearDNA unknown 11 CCCTCTAGAG TGGACACCAG CTGTAGCTGT TTCTTCCGAG GAGCG 45 24base pairs nucleic acid single linear DNA unknown 12 GTGCAGCCCATGGCCCTGAT TGTG 24 36 base pairs nucleic acid single linear DNA unknown13 TTCATTGGGC TAGGCATCTT CTTCAGATCT AGGTGC 36 33 base pairs nucleic acidsingle linear DNA unknown 14 TTCTTCAGAT CTAGGCGCAG TTTTATTGAT GAA 33 30base pairs nucleic acid single linear DNA unknown 15 CGTAAAACGCGTTTAAGGCA TTTTCTTTTC 30 33 base pairs nucleic acid single linear DNAunknown 16 GGGGTTCTAG AGACATTGTG ATATCMCARW CTC 33 60 base pairs nucleicacid single linear DNA unknown 17 CTGATAAGAT TTAGATTCGG AGCCAGAACCGGAAGATTTA CCTTCTGCAG CATCAGCCCG 60 34 base pairs nucleic acid singlelinear DNA unknown 18 GGGGTTCTAG AGAGGTCCAG CTGCARCARY CTGG 34 47 basepairs nucleic acid single linear DNA unknown 19 GGCTCCGAAT CTAAATCTTATCAGGAGGTC CAGCTGCARC ARYCTGG 47 27 base pairs nucleic acid singlelinear DNA unknown 20 ATAGACCATG GGGGCTGTTG TTTTGGC 27 30 base pairsnucleic acid single linear DNA unknown 21 ATAGAACGCG TGTCAGGCTGTTGTTTTGGC 30 24 amino acids amino acid linear peptide unknown 22 MetAsp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15Leu Pro Gly Ala Lys Cys Ser Arg 20 21 amino acids amino acid linearpeptide unknown 23 Met Gly Trp Ser Cys Lys Arg Arg Ser Ser Glu Glu ThrAla Thr Ala 1 5 10 15 Gly Val His Ser Arg 20 110 amino acids amino acidlinear peptide unknown 24 Asp Ile Val Ile Ser Gln Ser Pro Ser Thr LeuAla Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Asn Cys Lys Ser SerGln Ser Leu Phe Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp PheGln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala SerThr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp LeuAla Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Tyr Leu Arg Thr Phe Gly GlyGly Thr Arg Leu Glu 100 105 110 28 amino acids amino acid linear peptideunknown 25 Arg Ala Asp Ala Ala Glu Gly Lys Ser Ser Gly Ser Gly Ser GluSer 1 5 10 15 Lys Ser Tyr Gln Gly Ser Glu Ser Lys Ser Tyr Gln 20 25 122amino acids amino acid linear peptide unknown 26 Glu Val Gln Leu Gln GlnSer Gly Ala Glu Leu Val Lys Ser Gly Ala 1 5 10 15 Ser Val Lys Leu SerCys Lys Ala Ser Gly Phe Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp ValLys Gln Arg Ala Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr ProGly Ser Asn Thr Ile Asn Tyr Ser Glu Asn Phe 50 55 60 Lys Lys Lys Ala ThrLeu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu SerSer Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu ArgLeu Leu Gly Phe Val Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr ValSer Thr Ala Lys Thr Thr Ala 115 120 35 amino acids amino acid linearpeptide unknown 27 Met Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu LeuLeu Phe Ile 1 5 10 15 Gly Leu Gly Ile Phe Phe Arg Ser Arg Arg Ser PheIle Asp Glu Lys 20 25 30 Lys Met Pro 35

What is claimed is:
 1. A method of immunizing an individual against atarget protein comprising administering to said individual a plasmidcomprising a nucleotide sequence that encodes an immunogenic targetprotein linked to or comprising an intracellular targeting sequence,operably linked to regulatory elements, wherein said intracellulartargeting sequence is DKQTLL (SEQ ID NO:1) or DEKKMP (SEQ ID NO:3) atthe C terminal of said immunogenic target protein.
 2. A method ofimmunizing an individual against a target protein comprisingadministering to said individual a plasmid comprising a nucleotidesequence encoding an immunogenic target protein linked to or comprisingan intracellular targeting sequence, operably linked to regulatoryelements, wherein said intracellular targeting sequence directs saidimmunogenic target protein to be secreted.
 3. The method of claim 2wherein said immunogenic target protein is an allergen, pathogenantigen, cancer-associated antigen or antigen linked to cells associatedwith autoimmune diseases, or can induce an immune response thatcross-reacts with an allergen, pathogen antigen, cancer-associatedantigen or antigen linked to cells associated with autoimmune diseases.4. The method of claim 2 wherein said immunogenic target protein is anallergen, pathogen antigen, cancer-associated antigen or antigen linkedto cells associated with autoimmune diseases.
 5. The method of claim 2wherein said intracellular targeting sequence is an N-terminalhydrophobic leader sequence or an immunoglobulin leader sequence.
 6. Amethod of immunizing an individual against a target protein comprisingadministering to said individual a plasmid comprising a nucleotidesequence encoding an immunogenic target protein linked to or comprisingan intracellular targeting sequence, operably linked to regulatoryelements, wherein said intracellular targeting sequence directs saidimmunogenic target protein to be secreted, wherein said intracellulartargeting sequence comprises SEQ ID NO:22.
 7. A method of immunizing anindividual against a target protein comprising administering to saidindividual a pharmaceutical composition comprising a plasmid having anucleotide sequence encoding an immunogenic target protein linked to orcomprising an intracellular targeting sequence, operably linked toregulatory elements, wherein said intracellular targeting sequencedirects said immunogenic target protein to be secreted.
 8. The method ofclaim 7 wherein said pharmaceutical composition further comprises apolynucleotide function enhancer.
 9. The method of claim 8 wherein saidpolynucleotide function enhancer is bupivacaine.
 10. A method ofimmunizing an individual against a target protein comprisingadministering to said individual a plasmid comprising a nucleotidesequence encoding an immunogenic target protein linked to or comprisingan intracellular targeting sequence, operably linked to regulatoryelements, wherein said intracellular targeting sequence directs saidimmunogenic target protein to be localized in the cytoplasm of a cell.11. The method of claim 10 wherein said immunogenic target protein is anallergen, pathogen antigen, cancer-associated antigen or antigen linkedto cells associated with autoimmune diseases, or can induce an immuneresponse that cross-reacts with an allergen, pathogen antigen,cancer-associated antigen or antigen linked to cells associated withautoimmune diseases.
 12. The method of claim 10 wherein said immunogenictarget protein is an allergen, pathogen antigen, cancer-associatedantigen or antigen linked to cells associated with autoimmune diseases.13. A method of immunizing an individual against a target proteincomprising administering to said individual a plasmid comprising anucleotide sequence encoding an immunogenic target protein linked to orcomprising an intracellular targeting sequence, operably linked toregulatory elements, wherein said intracellular targeting sequencedirects said immunogenic target protein to be localized in the cytoplasmof a cell, wherein said intracellular targeting sequence comprises SEQID NO:23.
 14. A method of immunizing an individual against a targetprotein comprising administering to said individual a pharmaceuticalcomposition comprising a plasmid having a nucleotide sequence encodingan immunogenic target protein linked to or comprising an intracellulartargeting sequence, operably linked to regulatory elements, wherein saidintracellular targeting sequence directs said immunogenic target proteinto be localized in the cytoplasm of a cell.
 15. The method of claim 14wherein said pharmaceutical composition further comprises apolynucleotide function enhancer.
 16. The method of claim 15 whereinsaid polynucleotide function enhancer is bupivacaine.
 17. A method ofimmunizing an individual against a target protein comprisingadministering to said individual a plasmid comprising a nucleotidesequence encoding an immunogenic target protein linked to or comprisingan intracellular targeting sequence, operably linked to regulatoryelements, wherein said intracellular targeting sequence directs saidimmunogenic target protein to be localized in the cell membrane of acell.
 18. The method of claim 17 wherein said immunogenic target proteinis an allergen, pathogen antigen, cancer-associated antigen or antigenlinked to cells associated with autoimmune diseases, or can induce animmune response that cross-reacts with an allergen, pathogen antigen,cancer-associated antigen or antigen linked to cells associated withautoimmune diseases.
 19. The method of claim 17 wherein said immunogenictarget protein is an allergen, pathogen antigen, cancer-associatedantigen or antigen linked to cells associated with autoimmune diseases.20. The method of claim 17 wherein said intracellular targeting sequencecomprises an N-terminal hydrophobic sequence and an internal hydrophobicregion.
 21. A method of immunizing an individual against a targetprotein comprising administering to said individual a pharmaceuticalcomposition comprising a plasmid having a nucleotide sequence encodingan immunogenic target protein linked to or comprising an intracellulartargeting sequence, operably linked to regulatory elements, wherein saidintracellular targeting sequence directs said immunogenic target proteinto be localized in the cell membrane of a cell.
 22. The method of claim21 wherein said pharmaceutical composition further comprises apolynucleotide function enhancer.
 23. The method of claim 22 whereinsaid polynucleotide function enhancer is bupivacaine.
 24. A method ofimmunizing an individual against a target protein comprisingadministering to said individual a plasmid comprising a nucleotidesequence encoding an immunogenic target protein linked to or comprisingan intracellular targeting sequence, operably linked to regulatoryelements, wherein said intracellular targeting sequence directs saidimmunogenic target protein to be localized in the endoplasmic reticulumof a cell, and wherein said immunogenic target protein is an allergen,pathogen antigen, cancer-associated antigen or antigen linked to cellsassociated with autoimmune diseases, or can induce an immune responsethat cross-reacts with an allergen, pathogen antigen, cancer-associatedantigen or antigen linked to cells associated with autoimmune diseases.25. The method of claim 24 wherein said immunogenic target protein is anallergen, pathogen antigen, cancer-associated antigen or antigen linkedto cells associated with autoimmune diseases.
 26. A method of immunizingan individual against a target protein comprising administering to saidindividual a plasmid comprising a nucleotide sequence encoding animmunogenic target protein linked to or comprising an intracellulartargeting sequence, operably linked to regulatory elements, wherein saidintracellular targeting sequence directs said immunogenic target proteinto be localized in the endoplasmic reticulum of a cell, and wherein saidimmunogenic target protein is an allergen, pathogen antigen,cancer-associated antigen or antigen linked to cells associated withautoimmune diseases, or can induce an immune response that cross-reactswith an allergen, pathogen antigen, cancer-associated antigen or antigenlinked to cells associated with autoimmune diseases, wherein saidintracellular targeting sequence comprises SEQ ID NO:3 at the C-terminusof said immunogenic target protein or SEQ ID NO:27.
 27. A method ofimmunizing an individual against a target protein comprisingadministering to said individual a pharmaceutical composition comprisinga plasmid having a nucleotide sequence encoding an immunogenic targetprotein linked to or comprising an intracellular targeting sequence,operably linked to regulatory elements, wherein said intracellulartargeting sequence directs said immunogenic target protein to belocalized in the endoplasmic reticulum of a cell, and wherein saidimmunogenic target protein is an allergen, pathogen antigen,cancer-associated antigen or antigen linked to cells associated withautoimmune diseases, or can induce an immune response that cross-reactswith an allergen, pathogen antigen, cancer-associated antigen or antigenlinked to cells associated with autoimmune diseases.
 28. The method ofclaim 27 wherein said pharmaceutical composition further comprises apolynucleotide function enhancer.
 29. The method of claim 28 whereinsaid polynucleotide function enhancer is bupivacaine.
 30. A method ofimmunizing an individual against a target protein comprisingadministering to said individual a plasmid comprising a nucleotidesequence encoding an immunogenic target protein linked to or comprisingan intracellular targeting sequence, operably linked to regulatoryelements, wherein said intracellular targeting sequence directs saidimmunogenic target protein to be localized in a lysosome of a cell, andwherein said intracellular targeting sequence comprises SEQ ID NO:1. 31.The method of claim 30 wherein said immunogenic target protein is anallergen, pathogen antigen, cancer-associated antigen or antigen linkedto cells associated with autoimmune diseases, or can induce an immuneresponse that cross-reacts with an allergen, pathogen antigen,cancer-associated antigen or antigen linked to cells associated withautoimmune diseases.
 32. The method of claim 30 wherein said immunogenictarget protein is an allergen, pathogen antigen, cancer-associatedantigen or antigen linked to cells associated with autoimmune diseases.33. A method of immunizing an individual against a target proteincomprising administering to said individual a pharmaceutical compositioncomprising a plasmid having a nucleotide sequence encoding animmunogenic target protein linked to or comprising an intracellulartargeting sequence, operably linked to regulatory elements, wherein saidintracellular targeting sequence directs said immunogenic target proteinto be localized in a lysosome of a cell, and wherein said intracellulartargeting sequence comprises SEQ ID NO:1.
 34. The method of claim 33wherein said pharmaceutical composition further comprises apolynucleotide function enhancer.
 35. The method of claim 34 whereinsaid polynucleotide function enhancer is bupivacaine.